The Coronavirus disease 2019 (COVID-19) pandemic was marked by an increase in diagnosis and treatment delays for a range of medical conditions. Yet the impact of the pandemic on the management of tick-borne diseases, which frequently manifest as an acute febrile illness similar to COVID-19, has not been well described.Rickettsiarickettsii or Ehrlichia indirect immunofluorescence assay immunoglobulin G antibody test result. Of the 897 patients who had an eligible diagnosis, 240 (26.8%) met the inclusion criteria. The main outcome was time from initial presentation to definitive diagnosis and treatment.In this retrospective cohort study of patients with suspected tick-borne disease attending the University of North Carolina Health facilities, we compared the timeliness of diagnosis and treatment in a “pre-COVID” period (March 2019 to February 2020) and a “post-COVID” period (March 2020 to February 2021). Participants included patients with an ICD-10 diagnosis code of spotted fever group rickettsiosis or ehrlichiosis and a positive P = 0.03), and for a treatment delay > 7 days, 1.65 . The odds of a delay in diagnosis were similar for patients in the post- and pre-COVID periods, with an aOR of 1.61  for delays > 0 days, and aOR of 1.72  for delays > 7 days.During the 2-year study period, 126 (52.5%) patients were grouped in the pre-COVID period and 114 (47.5%) were grouped in the post-COVID period; 120 (50.0%) were female; and 139 (57.9%) were aged > 50 years. Comparing the post-COVID to the pre-COVID period, the adjusted odds ratio (aOR) for delay in treatment > 0 days was 1.81 (95% confidence interval [CI] 1.07–3.07, The odds of a delay in treatment > 0 days were significantly higher in the post-COVID period than in the pre-COVID period. However, the odds of a delay in treatment > 7 days, or a delay in diagnosis, were similar between these two periods. Shifts in care-seeking, alternative care delivery models and prioritization of COVID-19 may contribute to diminished timeliness of treatment for patients with tick-borne diseases.The online version contains supplementary material available at 10.1186/s13071-023-05917-8. Notably, the annual number of TBD cases doubled over the period of analysis with year-over-year increases in Lyme disease, ehrlichiosis and spotted fever group rickettsiosis (SFGR) [Amblyommaamericanum) is prevalent. This tick is a vector of Ehrlichiachaffeensis and Ehrlichiaewingii, both of which cause acute febrile illness that may progress to more severe and ultimately fatal disease within 7 to 14 days [Rickettsiarickettsii, is the most fatal tick-borne illness in the USA [While less frequently reported than Lyme disease, tick-borne rickettsiosis and ehrlichiosis are increasingly important public health threats , particu 14 days . Rocky M the USA . Yet, th the USA , 10.The global emergence of the coronavirus disease 2019 (COVID-19) pandemic, with the first cases emerging in late 2019, caused substantial changes in care-seeking behaviors and access to health services. Notable changes affecting patients included deferral of acute and routine care, limited options to receive in-person evaluations and greater use of telemedicine, compounded by the requirement to test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prior to hospitalization. A number of studies have demonstrated how these changes contributed to misdiagnosis and delayed care and ultimately led to worse patient outcomes across a range of conditions –15. The Very little is known about the impact of the COVID-19 pandemic on the diagnosis and treatment of TBDs. In one small study, three patients with TBD experienced an increased duration of time between symptom presentation and diagnosis due to fears and restrictions related to the pandemic . SimilarNorth Carolina (NC) experiences some of the highest incidence rates of SFGR and ehrlichiosis in the USA, often accounting for close to 16% and 9% of national totals reported to the CDC, respectively . The staR.rickettsii or Ehrlichia indirect fluorescent immunoglobulin G (IgG) antibody test result , both of which exhibit substantial cross-reactivity across species (i.e. R.rickettsii and R.parkeri) [Ehrlichia is available at UNC as a sendout to Mayo Clinic Laboratories but is not routinely ordered. Furthermore, PCR cannot be performed on stored serum (retrospective testing) [Patients with suspected SFGR or ehrlichiosis were identified utilizing the Carolina Data Warehouse for Health, a central repository containing clinical, research and administrative data sourced from University of North Carolina (UNC) Health, the largest academic health system in the state of North Carolina. In 2021, UNC Health reported 3.5 million clinical visits and 470,000 Emergency Department visits. Our study population included patients with an International Classification of Diseases, Tenth Revision (ICD-10) diagnosis code of SFGR (A77.0) or ehrlichiosis (A77.40) who were seen at any UNC Health facility utilizing Epic as the electronic medical record system in order to facilitate chart abstraction. Eligible periods included March 2019 to February 2020 (herein “pre-COVID”) and March 2020 to February 2021 (herein “post-COVID”). The post-COVID period approximates the first year of the pandemic as declared by the WHO on 11 March 2020 [parkeri) . PCR testesting) .Rickettsiaamblyommatis and E.ewingii, and high seroprevalence of antibodies reactive with R.rickettsii and E.chaffeensis [R.rickettsii and ≥ 1:64 for E.chaffeensis) and a more specific definition of probable cases than the CDC definition. For each eligible patient, we abstracted demographic and clinical information, including the results of laboratory testing. In addition, we documented: (i) the time in days from initial presentation to definitive diagnosis and treatment; (ii) the use of telehealth visits, if applicable; and (iii) the results of any severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing performed as part of the evaluation of symptoms. We also documented hospitalizations, intensive care unit (ICU) admissions, psychiatric referrals , sepsis and deaths during the course of a confirmed tick-borne illness.Each MRN was reviewed to confirm that the corresponding individual met specific inclusion criteria Table . These cffeensis , 23, we χ2) to test the hypothesis of no association between COVID period and sex, age group, health facility and insurance status. We also conducted Fisher’s exact tests to determine whether there was an association between pre- and post-COVID periods and each of five serious outcomes: hospitalization, sepsis, ICU admission, psychiatric referrals and death. All information was recorded in a case report  and then again defining it as > 7 days, which has been shown to be associated with serious clinical outcomes . AdditioWe employed logistic regression to evaluate the relationship between the two periods and each of our primary outcomes. These outcomes were dichotomized in two ways: (i) if the time was > 0 days; and (ii) if the time was > 7 days. All models were adjusted for age group (e.g. < 50 vs ≥ 50 years) and sex as these variables were hypothesized to confound the association of interest. Odds ratios (OR) were calculated for delays, stratified by COVID period, age and sex, while adjusting for all other variables in the model. All analyses were conducted using SAS Studio 3.8 . All figures were created using R 4.0.5 .A total 897 patients had an eligible ICD diagnosis, of whom 240 (26.8%) met the inclusion criteria, including 126 (52.5%) in the pre-COVID period and 114 (47.5%) in the post-COVID period. Almost all of the excluded patients reported fewer than two symptoms. Of the 240 patients who met inclusion criteria, 14 (5.8%) had at least a fourfold increase in titer between acute and convalescent serologies; 124 (51.7%) had symptoms present for ≤ 14 days and only an acute sample with a titer ≥ 1:256; and 102 (42.5%) were treated empirically, even if symptoms were present for ≥ 14 days. The number of patients in each category was similar in the two periods.N = 120, 50.0%), but trended older with a majority of patients aged > 50 years. The two groups were generally similar across sex, age, season of presentation and health insurance status. While outpatient visits were the most common care setting in both timeframes (52.4% pre-COVID and 57.5% post-COVID), more participants in the pre-COVID period  were seen in an Emergency Department compared to the post-COVID period . Telemedicine was only utilized by participants in the post-COVID timeframe, accounting for 11.4% (N = 13) of initial visits. No statistically significant associations were detected between pandemic group and sex, age category, health facility, duration of symptoms at first visit or insurance status. In the post-COVID period, 29 (25.4%) patients underwent one SARS-CoV-2 test while 11 (9.6%) underwent two tests; however, only three (7.0%) tests were positive for SARS-CoV-2  (Table P = 0.01). When restricting the analyses to either delays > 0 days or delays > 7 days, however, the differences were not statistically significant. In the pre-COVID period, 37 out of 128 patients (28.9%) had delays in diagnosis > 7 days, and 42 out of 135 patients (31.1%) had delays in treatment > 7 days. In the post-COVID period, 47 out of 105 (44.8%) patients had delays in diagnosis > 7 days, while 49 out of 111 (44.1%) patients had delays in treatment > 7 days. More people in the pre-COVID period, however, received diagnosis and treatment on the day of initial presentation (i.e. delay of 0 days) than in the post-COVID period. Density plots reveal differences in time to both outcomes between the two periods  day, while in the post-COVID period the median time was 5.0  days (P = 0.03) and 1.65  for a treatment delay > 7 days. The odds of a delay in TBD diagnosis were similar when comparing the post- to pre-COVID periods, including aOR = 1.61  for delays > 0 days and aOR = 1.72  for delays > 7 days.Logistic regression modeling showed an increase in the odds of a delay in treatment for the post-COVID period compared to the pre-COVID period Table . CompariIn the pre-COVID period there were 19 (15.1%) hospitalizations and two (1.6%) deaths associated with TBD compared to 16 14.0%) hospitalizations and zero deaths in the post-COVID period . Given the heightened awareness of COVID-19 during the study period, providers may have anchored on a COVID-19 diagnosis on initial presentation, delaying the consideration of TBD and empirical treatment while SARS-CoV-2 testing was performed. We recommend that providers broaden their differential in a patient with acute febrile illness, and to suspect SFGR or ehrlichiosis in the setting of relevant epidemiologic exposure, tick season, typical clinical manifestations and laboratory test abnormalities , 28.Furthermore, decreased access to outpatient clinics, hesitation about going to the Emergency Department due to long waits and fears of contracting SARS-CoV-2 and increasing utilization of telemedicine appointments may have negatively impacted the quality of care . TelemedGiven the risk of rapid progression, it was reassuring that a large proportion of patients received a diagnosis and/or treatment at initial presentation . Only 14R.parkeri, which has serologic cross-reactivity with R.rickettsii [The fact that we did not observe any differences in important outcomes, including hospitalization, ICU admission or death, partly reflects the relative infrequency of these events. Some of this infrequency may be attributable to infection with less pathogenic rickettsial pathogens such as ckettsii . AdditioTo our knowledge, this study is the largest and most robust analysis of potential impacts of the COVID-19 pandemic on the management of TBD. Our study has a number of strengths, including the use of a high-quality database, rigorous eligibility criteria, access to the complete medical record and location in North Carolina, where rates of SFGR and ehrlichiosis are among the highest in the nation. The study also has several limitations. First, while ours is the largest study to date, the sample size was still relatively small, especially in regard to our analysis of serious outcomes. Second, the study population was drawn from patients seen at a large academic medical system. Therefore, patient care experience may not reflect that of other health facilities, such as community hospitals or private practices that were more severely impacted by the pandemic. Lastly, our study cohort skewed towards older patients, who may have been more likely to develop overt clinical symptoms or have more health concerns related to age, given that this was an established and well-published risk factor for severe COVID-19. However, we note that the cohort looks demographically similar to that of our previous studies conducted prior to the pandemic .Future research directions include comparing pre-pandemic data to data from later in the pandemic (e.g. 2022–2023) to assess for a reversion to pre-pandemic care patterns, assessing the effect of the pandemic on TBDs endemic to other geographical regions, such as Lyme disease, investigating delays in diagnosis and treatment of TBDs in non-academic settings and using PCR-based testing to diagnose SFGR and ehrlichiosis.Our study found delays in the diagnosis and treatment of SFGR and ehrlichiosis during the COVID-19 pandemic compared to the pre-COVID period. Key factors driving these delays include a shift in care-seeking, care delivery models and prioritization of COVID-19 as a potential cause of illness. These concerning findings highlight the effect of COVID-19 on the diagnosis and treatment of TBDs and merit both further study and educational interventions to minimize preventable morbidity and mortality attributable to TBD.Additional file 1: Table S1. Results of serological testing for SARS-CoV-2, R.rickettsii and Ehrlichia. Figure S1. Case report form for health system activities and patient outcomes.


Lagerstroemia speciosa (L.) Pers. flower buds and their efficacy against antimicrobial and anticancer activities. The UV-Visible spectrum was found at 413 nm showing a typical resonance spectrum for L. speciosa flower bud extract-assisted silver nanoparticles (Ls-AgNPs). Fourier transform infrared analysis revealed the presence of amines, halides, and halogen compounds, which were involved in the reduction and capping agent of AgNP formation. X-ray diffraction analysis revealed the face-centered cubic crystals of NPs. Energy dispersive X-ray verified the weight of 39.80% of silver (Ag), TEM analysis revealed the particles were spherical with a 10.27 to 62.5 nm range, and dynamic light scattering recorded the average particle size around 58.5 nm. Zeta potential showed a significant value at −39.4 mV, and finally, thermo-gravimetric analysis reported higher thermal stability of Ls-AgNPs. Further, the obtained Ls-AgNPs displayed good antimicrobial activity against clinical pathogens. In addition, a dose-dependent decrease in the anticancer activity by MTT assay on the osteosarcoma (MG-63) cell line showed a decrease in the cell viability with increasing in the concentration of Ls-AgNPs with an IC50 value of 37.57 µg/mL. Subsequently, an apoptotic/necrosis study was conducted with the help of Annexin-V/PI assay, and the results indicated a significant rise in early and late apoptosis cell populations. Therefore, green synthesized Ls-AgNPs were found to have potent antimicrobial and anticancer properties making them fascinating choices for future bio-medical implementations.Biosynthesized nano-composites, such as silver nanoparticles (AgNPs), can be engineered to function as smart nano-biomedicine platforms for the detection and management of diverse ailments, such as infectious diseases and cancer. This study determined the eco-friendly fabrication of silver nanoparticles using  Nanotechnology research will be at the forefront in the 21st century; it is a novel and developing technology with broad applications. It includes synthesizing new materials and their use in various fields with dimensions ranging from 1 to 100 nanometers, which could significantly change their physico-chemical properties. Several materials are used to make nanoparticles, and the chemical composition, size, and form can affect the particle’s characteristics . SeveralUtilizing plant extracts to synthesize silver nanoparticles is a highly efficient, economically viable, and bio-sustainable approach that circumvents hazardous chemicals. In recent years, numerous bio-sustainable methods for the expeditious production of silver nanoparticles have been documented, which employ aqueous extracts of various plant components, such as leaves, bark, flowers, and roots . The useLagerstroemia speciosa (L.) Pers. is a flowering tree in the Lythraceae family. It is indigenous to Southeast Asia, primarily the Philippines, but has been introduced and produced in various tropical and subtropical climes worldwide. It can help with diabetic treatment. The tree’s leaves contain bioactive substances, such as corosolic acid, which have been found to promote insulin function and increase glucose uptake by cells. It has excellent antioxidant properties due to many phenolic components, such as ellagitannins, flavonoids, and anthocyanins. Anti-inflammatory characteristics may help reduce inflammation and treat conditions, such as rheumatism and intestinal inflammation, as well as antibacterial activity. In traditional medicine, it is utilized for a range of additional uses. It is a diuretic, an antihypertensive, and an antipyretic. Furthermore, the tree’s bark, roots, and leaves are used to treat diarrhea, skin diseases, and respiratory problems [problems ,11.Cancer is presently the most lethal disease characterized by unregulated cellular proliferation and growth, which poses a significant challenge when it metastasizes and becomes refractory to conventional therapeutic interventions . Among tSome researchers have also been employed to assess the efficacy of novel drugs for treating osteosarcoma and other conditions affecting the bones ,15,16,17L. speciosa provide interest in the present investigation to synthesize silver nanoparticles.In addition, nanotechnology is still a good topic that can contribute significantly to innovation and advancement in the scientific community. The advancement of numerous fields and overcoming the difficulties posed by operating at the nano-scale will probably be driven by ongoing research in the future. Overall, the medicinal properties of the plant Objectives of the research:Lagerstroemia speciosa flower bud extract, their characterizations using different analytical tools, and the evaluation of their biological activities. Therefore, the following objectives were carried out using L. speciosa flower bud extract:(i)L. speciosa plant;Bio-inspired synthesis of AgNPs utilizing the flower bud extract of the (ii)Characterization of biosynthesized AgNPs by using various analytical techniques;(iii)Evaluating the antimicrobial activity of biosynthesized AgNPs against human clinical pathogens;(iv)Determination of in vitro anticancer potential by MTT assay using biosynthesized AgNPs against MG-63 cell line and exploring the apoptotic inducing ability of biosynthesized AgNPs through flow cytometry.This research aims to synthesize AgNPs from L. speciosa flower buds were collected from the Botanical garden on the campus of Karnatak University, Dharwad, Karnataka, India  (′ 57″ E) A,B. The 3 was minced in 1000 mL of distilled water in an Erlenmeyer flask to obtain a 1 mM solution. Obtained flower bud extract (100 mL) was mixed with 1 mM AgNO3 solution (800 mL) in the ratio 1:8 (v/v) in a clean conical flask, and the pH was set to 9.0 and kept at room temperature in the dark for 24 h. The color of the reaction solution changed after the incubation period was the first hint of the fabrication of Ls-AgNPs. Ls-AgNPs were obtained from the reaction solution using subsequent centrifugation at 10,000 rpm for 20 min; then, air-dried and stored for further investigations [Approximately 0.1689 g of AgNOUsing a UV-Visible spectrophotometer , the spectroscopic analysis was utilized to monitor the formation of Ls-AgNPs. The reaction mixture was placed in a quartz cuvette, and the absorbance from 300 to 600 nm was measured. The graph was made by plotting wavelength against absorbance .L. speciosa flower bud extract and Ls-AgNPs separately with 10% KBr. The spectra of the flower bud extract and Ls-AgNPs were recorded using an FTIR spectrophotometer  over a wavelength range of 4000 to 400 cm−1 [The detection of functional groups serving as stabilizing and capping agents during the synthesis of Ls-AgNPs was accomplished through FTIR analysis. The Ls-AgNPs were subjected to a drying process at 45 °C for a duration of 24 h in order to eliminate any water molecules present. Thin pellets were prepared by combining 400 cm−1 .α radiation (λ = 1.54056 A°) source. The spectrum was recorded in the 2θ scanning range of 30° to 90°, and the spectrum was matched with the standard JCPDS file no: 04-0783 [XRD analysis was accomplished to determine the crystalline nature, phase variation, and grain size of Ls-AgNPs. The Ls-AgNPs were oven-dried at 50 °C and analyzed by an XRD analyzer  employing a Cu-K 04-0783 .For this analysis, the Ls-AgNPs were utterly dried, and a small amount of sample was mounted on carbon tape and coated with gold sputtering for 2 min. It was then analyzed with the instrument  to identify the elemental makeup of Ls-AgNPs .To figure out the size, shape, and structures of Ls-AgNPs, TEM  analysis was performed. Five microliters of Ls-AgNPs were placed on the surface of the copper grid and dried in desiccation for 48 h. Later, the Ls-AgNPs were scanned at a voltage of 300 keV, and images were captured at the resolution of 7000× to 8000× .The surface charge of Ls-AgNPs was ascertained by zeta potential analysis in a colloidal system. A uniform suspension of nanoparticles was prepared by ultra-sonication and centrifuged at 6000 rpm for 20 min. It was then analyzed by a Nano analyzer  at 3.4 eV. The dispersal type and average size of Ls-AgNPs were analyzed with DLS measurement .The thermal properties and decomposition of Ls-AgNPs synthesized through the biological process were analyzed using a TGA instrument . A predetermined amount of Ls-AgNPs was introduced into the furnace at ambient temperature (approximately 27 °C) and subjected to a gradual temperature increase up to 800 °C, with a heating rate of 10 °C/min, In contrast, inert nitrogen gas (at a flow rate of 40 mL/min) was passed over the sample, as described in references ,29.Staphylococcus aureus (MTCC 6908), Gram-negative organism: Escherichia coli (MTCC 40), yeast: Candida albicans (MTCC 227) and Candida glabrata (MTCC 3019) were procured from IMTECH, Chandigarh, India. A suspension of Ls-AgNPs (1 mg/mL) was prepared using DMSO, and nutrient agar medium was utilized for antimicrobial activity. One hundred microliters of bacterial and yeast inoculums (0.5 McFarland concentrations) were swabbed on medium surfaces using sterile cotton swabs. Sterile cork borers were utilized to create wells measuring 6 mm in diameter. The wells were filled with varying volumes of Ls-AgNPs, precisely 25, 50, 75, and 100 µL. Distilled water served as the negative control; streptomycin and nystatin (1 mg/mL) were chosen as the standard reference for bacteria and yeast, respectively. After 18 h of incubation period at 37 °C, the diameter of the zone of inhibition surrounding each well was measured in millimeters (mm) [The antimicrobial efficacy of Ls-AgNPs was determined by employing the agar well diffusion technique against four distinct microbial pathogens. The Gram-positive organism: ata MTCC 19 were pers (mm) .2. The MTT assay was conducted using a negative control of medium and cells without Ls-AgNPs. Quercetin (30 µM/mL) was utilized as a positive control. MG-63 cells were seeded at a density of approximately 20,000 cells per well in 96-well plates and incubated for 24 h to allow for growth. Subsequently, cellular entities were subjected to a suspension of AgNPs, with concentrations varying from 12.5 to 200 µg/mL, along with the positive control, for 24 h. Following incubation, the medium was decanted and replaced with 200 µL of MTT reagent per well. The cells were then kept for 3 h of incubation. MTT-formazan crystals were dissolved by adding 100 µL of DMSO and gently shaking the mixture. The absorbance of the resulting suspension was recorded at 570 nm using an ELISA reader  [50) was calculated: Y = Mx + c. The percentage of cell viability was determined using the below-following formula:The MG-63 cell line, derived from human osteosarcoma, was procured from NCCS, Pune. The anti-proliferative assay was conducted using the MTT-96 well plate method. The cell line was subjected to sub-culturing in DMEM and added with FBS (10%). The cancer cells were maintained at 37 °C for 24 h in an atmosphere containing 5% COVT, USA) ,33, whic50 concentration of 37.57 µg/mL for 24 h under controlled conditions of 37 °C and 5% CO2. The cells without any treatment were designated as untreated control, while quercetin-treated control cells were designated as such. Further, the cancer cells were washed using PBS buffer and subjected to a 3–4 min treatment with 200 µL of trypsin-EDTA at 37 °C. Subsequently, a volume of 2 mL of culture medium was introduced, and the cells were subjected to centrifugation at 300 rpm for 5 min, after which the supernatant was carefully removed. Following that, PBS was used to wash the cells twice, after which they were subjected to staining with 5 µL of Annexin-V and incubated in a light-free environment for 15 min. In addition, a mixture comprising five µL of PI and 1X binding buffer (400 µL) was cautiously vortexed with the cells. Subsequently, the cells were subjected to analysis via a flow cytometer , and the investigation outcomes were computed utilizing BD Cell Quest Pro Ver.6.0 software [The apoptotic effects of Ls-AgNPs on MG-63 cells were evaluated through the utilization of the FITC Annexin-V (51-65874X) and propidium iodide (PI) (51-66211E) labeling technique. A total of 20,000 MG-63 cancer cells were seeded into a 96-well plate and treated with Ls-AgNPs at the ICsoftware .n = 3), and the mean of three parallel experiments used to determine each value. The presentation of each outcome was in the form of mean ± standard deviation (±SD). The obtained results were subjected to statistical analysis using SPSS and Origin 2022 Pro version.The experiments were conducted in triplicate (3) suspension in a ratio of 1:8 at pH 9.0 to synthesize Ls-AgNPs. After 24 h of the incubation period, the reaction mixture turned dark brownish from pale yellow, indicating proper synthesis and fabrication of Ls-AgNPs (+) were bio-reduced into elemental silver (Ag°) and capped and stabilized. Synthesized Ls-AgNPs produced a characteristic absorption spectrum at 413 nm, suggesting metallic silver’s surface plasmon resonance (SPR) range (The aqueous flower bud extract of Ls-AgNPs was minced with one mM silver nitrate (AgNOLs-AgNPs A–C. Due R) range D.L. speciosa flower bud extract in the region of 4000 to 400 cm−1 , as depicted in 50 concentration of 37.57 µg/mL for a duration of 24 h. The treated cells exhibited a substantial increase in the population of early apoptosis cells (8.98%) and late apoptosis cells (39.8%), while the untreated cells did not exhibit any significant apoptosis. A test was performed to assess the occurrence of apoptosis/necrosis in the MG-63 cell line following treatment with a synthesized Ls-AgNPs solution at the ICL. speciosa flower bud extract with the determination of antimicrobial and anticancer activity. In the recent evolution of nanotechnology, many approaches were looked into synthesizing AgNPs from different parts of plant-like roots, leaves, stems, flowers, fruits, etc. The initial hint of Ls-AgNPs biosynthesis was assured by a color change in the reaction solution of flower bud extract and AgNO3 (1:8 v/v). The color change from pale yellowish to dark brownish indicated the surface plasmon resonance of metallic silver, which in turn attributed to the biosynthesis of Ls-AgNPs. During the synthesis, various phyto-constituents in the flower bud extract were directly proportional to the synthesis of Ls-AgNPs due to their reducing and capping activity [R. serrata flower bud extracts containing various phytochemicals in the reduction, capping, and stabilization of AgNPs [The present research reported the nano-bio-fabrication of silver nanoparticles using activity ,36. The of AgNPs .L. speciosa phytoconstituents was investigated by FTIR analysis. The shifting of peaks in the Ls-AgNPs FTIR spectrum was owing to the interaction between several chemical compounds and Ag+ [L. speciosa flower bud extract was encapsulated by phytochemicals and some acids due to functional groups, including alkanes, alkenes, amines, alcohols, aldehydes, ketones, etc. The functional groups on Ls-AgNPs showed the existence of primary and secondary amines, indicating that proteins were involved in the reduction and stabilization of Ag+. Thus, the AgNPs were reduced and stabilized by the L. speciosa flower extract’s nano-capping. This result was agreed with the report of Rajesh Kumar et al. [C. guianensis Aubl., bud extract expressed the presence of several functional groups.The molecular atmosphere on the surface of Ls-AgNPs due to the capping of  and Ag+ . The L. r et al. , where tCucumis sativus var. hardwickii exhibited a similar XRD pattern, having Bragg’s reflection indexes of 111, 200, 220, and 311 at 2θ angles of 38.25°, 46.41°, 64.55°, and 77.48°. According to the previous report, the additional peaks that were acquired could potentially be attributed to alternative crystalline phases associated with the inorganic components of the plant extract, which are present on the surface of the synthesized silver nanoparticles [The face-centered cubic crystalline nature of Ls-AgNPs was affirmed by X-ray diffractometry. The XRD pattern showed the reflection peaks at 38.17°, 44.34°, 64.50°, and 77.43° at 2θ degrees corresponding to the 111, 200, 220, and 311 facets of Bragg’s planes. Accordingly, a previous study involved in the synthesis of AgNPs from articles .+ ions to Ago by generating a major peak at 3 keV. The EDX spectrum also revealed the existence of other metals, such as carbon, chlorine, sodium, potassium, etc. Similarly, the AgNPs obtained from Syzygium aromaticum (Clove) extract demonstrated a strong signal for Ag with other peaks for calcium, carbon, oxygen, chlorine, potassium, etc. The elemental peaks, apart from Ag, were observed due to the presence of bio-molecules on the surface of AgNPs involved in the capping and stabilization; the peaks for carbon and oxygen might be due to the plant materials and atmospheric moisture [The EDX spectrum of Ls-AgNPs exhibited 39.80% of Ag in the sample. This also demonstrated the reduction of Agmoisture .C. guianensis Aubl. bud extract were spherical, 5 to 40 nm in size, and poly-dispersed [The TEM micrograph illustrated the shape of Ls-AgNPs as spherical and poly-dispersed with slight agglomerations at some points. The particle sizes were assessed in the range of 10.27 to 62.5 nm. Similar observations were recorded in previous studies, where the AgNPs synthesized from clove buds extract showed spherical-shaped, poly-dispersed AgNPs in size ranges of 10 to 50 nm. The report suggested that most AgNPs were aggregated, while a few were scattered with varied sizes . In otheispersed .H. tuberculatum extract was −42.6 mV, suggesting higher repulsion among AgNPs and their dispersion stability. According to previous studies, zeta potential higher than +30 mV or −30 mV was strongly cationic and strongly anionic; the more considerable negative/positive zeta potential value indicated higher repulsion among AgNPs, leading to higher stability [Zeta potential exhibits the nanoparticles’ dispersion stability and surface electric charge. The zeta analysis of Ls-AgNPs showed a sharp peak at −39.4 mV, indicating an anionic surface charge and moderate repulsion between AgNPs. The zeta potential value indicated moderate stability. Similarly, El-Aswar et al.  reportedtability .P. murex leaf extract AgNPs at 73.14 nm [The DLS analysis determined the size distribution profile of Ls-AgNPs and calculated the average size of particles as 58.5 nm. The prominent DLS spectrum was a sign of proper synthesis of nanoparticles. Brownian motion of nanoparticles in the suspension was the key to measuring the size of Ls-AgNPs. These results were in accordance with the previous report suggesting the calculated average size of 73.14 nm .C. pulcherrima aqueous flower-extract, where the AgNPs weight loss was observed constantly from 0 to 800 °C, and the total weight loss up to the end (800 °C) for the biosynthesized silver nanoparticles is nearly 71.68% [The TGA analysis illustrated the thermal stability of Ls-AgNPs by depicting the % weight loss at a range of temperatures from room temperature to 800 °C. Initially, at room temperature, the weight of Ls-AgNPs was 100%, gradually decreasing to 55% at 700 °C with a total weight loss of 46.25%. These results were compared with TGA results of synthesized NPs from y 71.68% .S. aureus exhibited greater susceptibility towards Ls-AgNPs, while E. coli demonstrated the highest resistance level compared to the other microbial pathogens selected. Singh [P. aeruginosa, E. coli, K. pneumonia, and B. subtilis. The findings indicate that the size of the dose administered may influence the efficacy of AgNPs against multidrug-resistant bacteria. Lee et al. [Tussilago farfara buds extracts for producing of AgNPs resulted in distinctive inhibition of the growth of various microbial strains. The fascinating antimicrobial efficacy can be attributed to the proportion of AgNPs’ extensive surface area to their volume, facilitating a more robust interaction with pathogens. The notable impact of AgNPs on microbial pathogens can be attributed to their diminutive size and the electrostatic force of attraction between the less negative to the positive charge of the nanoparticles and the negatively charged membrane of the microbial cells. The conversion of chemical and physiological properties has been observed to disrupt their normal physiological functions, such as permeability and respiration [The synthesized green Ls-AgNPs demonstrated antimicrobial activity that was dependent on the dosage. The findings of the current investigation indicate that d. Singh  reportede et al.  elucidatpiration ,49. Silvpiration .50 value of Ls-AgNPs was determined to be 37.57 µg/mL, indicating a promising outcome. An array of literature is available documenting the anti-proliferative properties of silver nanoparticles against diverse cancer cell lines. Vitrus vinifera fruit extract resulted in the inhibition of MG-63 cell proliferation. The observed cell inhibition was 55% at a concentration of 1.5 mM of AgNPs. According to Nayak et al. [The Ls-AgNPs that were bio-fabricated demonstrated remarkable anti-cancer properties on the MG-63 cell line, with the effect being dependent on the dosage administered. The experiment results demonstrate that the application of escalating concentrations of Ls-AgNPs (ranging from 12.5 to 200 µg/mL) to cancer cells resulted in a notable reduction in viable cells, decreasing from 85.78% to 6.87%. The untreated control group exhibited complete cell viability, while the standard group demonstrated cell viability of 30.92%. The ICk et al. , silver 50 value of 37.57 µg/mL. The primary mechanism of numerous anticancer drugs is to induce cancer cell death by triggering the apoptotic signaling pathways [2O3 nanoparticles at a concentration of 0.102 μg/mL resulted in an inevitable outcome. The quantification of cells with compromised membrane integrity was performed using PI/FITC uptake. The MG-63 cells subjected to Tb2O3 NPs exhibited a concentration-dependent uptake of PI/FITC and demonstrated early apoptosis rates of 5.3% and 6.4%, respectively. The study utilizing Annexin V-FITC/PI aimed to identify cells undergoing early and late stages of apoptosis. According to a previous study [The Annexin V-FITC/PI apoptosis method was utilized to verify the occurrence of apoptosis. The Ls-AgNPs exhibited a notable ability to induce apoptosis against the MG-63 cell line, with an ICpathways . Comparapathways  found thpathways  revealedus study , cells wus study . The obsus study ,56.L. speciosa flower buds. The confirmation of AgNP formation is supported by the UV-Vis spectrum, which displays a distinctive peak at 413 nm. The crystallinity of Ls-AgNPs was confirmed to be of the FCC type through XRD analysis. The stability of the Ls-AgNPs was demonstrated by the notable zeta potential value, while the average size of the particles was determined to be 58.5 nm through DLS analysis. The analysis conducted using TEM confirmed the presence of spherical particles that were randomly distributed, while the thermal stability of Ls-AgNPs was established through TGA analysis. Comprehending the influence of nanoparticles’ physical and chemical properties on their biological effects is of utmost importance. The Ls-AgNPs that were synthesized exhibit antimicrobial properties against various pathogens and demonstrate anticancer activity against the MG-63 cell line. Moreover, the flow cytometry technique was employed to evaluate the apoptosis/necrosis of neoplastic cells. The Annexin-V-FITC assay was employed to verify the induction of apoptosis initiation by Ls-AgNPs. This assay is widely utilized to distinguish living cells from those undergoing early and late apoptosis. The discernible actions exhibited by Ls-AgNPs are of significant importance in the advancement of novel therapeutic agents for the diagnosis of cancer. The employment of plant-mediated synthesis is deemed more advantageous compared to other methods due to its faster and more stable rate of metal ion reduction. Silver nanoparticles exhibit promising potential in biomedical applications, particularly in disease diagnosis, cellular and deep tissue imaging, drug/gene delivery, and multi-functional therapies.The present investigation delineated a facile, sustainable, and economical approach for the bio-fabrication of AgNPs utilizing the extract of


In an effort to providethe first accurate structural and spectroscopiccharacterization of the quasi-linear chain HONCO in its electronicground state, state-of-the-art computational approaches mainly basedon coupled-cluster (CC) theory have been employed. Equilibrium geometrieshave been calculated by means of a composite scheme based on CC calculationsthat incorporates up to the quadruple excitations and accounts forthe extrapolation to the complete basis set limit and core correlationeffects. This approach is proven to provide molecular structures withan accuracy better than 0.001 Å and 0.05° for bond lengthsand angles, respectively. Incorporation of vibrational effects permitsthis level of theory to predict rotational constants with an estimatedaccuracy of 0.1% or better. Vibrational fundamental bands have beenevaluated by means of a hybrid scheme based on harmonic frequenciescomputed using the CC singles, doubles, and a perturbative treatmentof the triples method (CCSD(T)) in conjunction with a quadruple-ζbasis set, with all electrons being correlated, and anharmonic correctionsfrom CCSD(T) calculations using a triple-ζ basis set, withinthe frozen-core approximation. Such a hybrid approach allowed us toobtain fundamental frequencies with a mean absolute error of about1%. To complete the spectroscopic characterization, vertical electronicexcitation energies have been calculated for the lowest singlet andtriplet states using the internally contracted multireference configurationinteraction (MRCI) method. Computations show that HONCO dissociatesinto OH + NCO upon the absorption of UV–vis light. In conclusion,we are confident that the highly accurate spectroscopic data providedherein can be useful for guiding future experimental investigationsand supporting the characterization of this molecule in atmosphericand astrophysical media, as well as in combustion. The relative energies of these isomerswere then established by accurate CCSD(T)/6-311G single-pointcomputations on top of B3LYP/6-311G optimized geometries, alsoincorporating zero-point-energy (ZPE) corrections. On the lowest singletB3LYP/6-311G PES, these authors identified 25 isomers and 50transition states, also including some weakly bound complexes. Amongthese systems, the HO−N=C=O molecularchain was found to be the lowest in energy. In a previous theoreticalinvestigation, Kulkarni and Koga,7 whostudied this molecule in connection with the NO + XCO reactions in the gas phase, concluded that HONCO, CH(=O)NO, and HNOCO,in order of increasing energy, correspond to the lowest-energy isomersat the same level of theory as above /6-311G energyon top of B3LYP/6-311G geometries).TheHONCO molecule and its isomers are important species that areinvolved in numerous chemical processes. For instance, they are implicatedin combustion of organic compounds and atmospheric reactions.ab initio study by Tourchiet al.8 of the stable forms of the pentatomic molecular system focused on their structural and vibrationalspectroscopy characterization.8 Twentyisomers of HONCO lying within the 0.0–5.7 eV energy windowwere identified, and confirmed the previous theoretical predictionsgiving the chain-like form as the most stable.8 Tourchi et al.8 performed structuraloptimizations at the CCSD(T) level in conjunction with the aug-cc-pVnZ  basis sets with the extrapolationto complete basis set (CBS) limit, followed by accurate predictionsof the fundamental bands. For the latter, a hybrid scheme combiningCCSD(T) harmonic frequencies with MP2 anharmonic corrections was employed.The principal focusof the above-cited theoretical works on HONCOisomers was to establish their role as key intermediates in reactionsthat are relevant for combustion and atmospheric processes, and avery limited theoretical investigation was instead devoted to theaccurate characterization of their spectroscopic properties. Differently,a more recent and thorough 12 more complex systemscontaining the NCO moiety, such as methyl isocyanate17 and ethyl isocyanate,9 have already beendiscovered. The observation of another element of the family wouldprovide an important step forward because little is known about thechemistry of isocyanates in space. The presence of the isocyanateradical (NCO)18 and N-protonated isocyanicacid (H2NCO+)19 in the ISMsuggests the possibility of incorporating the NCO moiety in moleculesof increasing complexity.The  family might have some relevance in astrochemistry,and, in particular, its most stable isomer HONCO is a potential interstellarspecies. Indeed, isocyanates (X−NCO) can be considered prebioticsystems because they play a role in the synthesis of amino acids,in the polymerization of peptides, and in the production of nucleotidesand nucleosides technique. Indeed, for the assignment of the high-resolution rotationalor infrared (IR) spectra, it is essential to determine molecular geometriesand vibrational frequencies at the highest possible level of accuracy.25 Most of these computations employ the CC method with singles, doubles,and a perturbative treatment of connected triple excitations26 (CCSD(T)). All calculations were performed inconjunction with the cc-pVnZ (n =Q-6) atomic basis sets of Dunning and co-workers.28Quantum-chemical calculationsfor the electronic ground state of HONCO were carried out with theCFOUR suite of quantum chemistry programs.30 and the geometry scheme32 approaches. The former, implemented in a black-box manner in theCFOUR program, has been exploited to perform geometry optimizationat the CCSD(T)/CBS+CV level, where CV denotes the incorporation ofcore-valence corrections. HF-SCF and CCSD(T) correlation energiesare extrapolated separately using Feller’s exponential33 (n = Q-6) and n–3  extrapolation formula,34 respectively. For CCSD(T), the frozen-core (fc)approximation is employed. The CV correction is evaluated by addingto the energy gradient the contribution corresponding to the differenceof all-electron (ae) and fc CCSD(T) energies, obtained with the cc-pCVQZbasis set.35 Overall, the CCSD(T)/CBS+CVenergy gradient is defined asBest-estimated structural parameters have been evaluated bycombining the gradient-schemer denotes a genericstructural parameter and rCBS+CV is thecorresponding value obtained from the CBS+CV geometry optimizationdefined by r(fT) correction is evaluated as the differencebetween r values computed at the fc-CCSDT/cc-pVTZand fc-CCSD(T)/cc-pVTZ levels, CCSDT standing for the CC singles,doubles, and triples method.36 The Δr(pQ) contribution is given by the difference between the r values computed at the fc-CCSDT(Q)/cc-pVDZ and fc-CCSDT/cc-pVDZlevels, with CCSDT(Q) being the CCSDT method augmented by a perturbativetreatment of quadruple excitations.37The geometry scheme has been employedto incorporatethe contributionsof the full treatment of triple excitations (fT) and the perturbativetreatment of quadruple excitations (pQ) in the cluster expansion,thus leading to the CCSD(T)/CBS+CV+fT+pQ structure:vide infra) mightalso be needed. According to vibrational perturbation theory to secondorder (VPT2),38 rotational constants consistof two terms: the equilibrium contribution, which is the dominantone 39 and the vibrational correction, which incorporatesthe effects of molecular vibration. For a generic vibrational state v, the expression of the corresponding rotational constant Bvi is given byBei isthe equilibrium rotational constant relative to the i-th inertial axis , and only depends on the equilibriumstructure (and isotopic mass composition). The second term is thevibrational correction, which is obtained by summing, over the vibrationalmodes r , the vibration–rotation interactionconstants (αri). If the vibrational groundstate is considered (B0i), the second term of Bvibi, which is—contraryto the single αri—devoid of anyresonance.40 The calculation of the vibration–rotationinteraction constants requires anharmonic force field computations,which were performed here at the fc-CCSD(T)/cc-pVTZ level. Additionally,the harmonic force field has been computed at the ae-CCSD(T)/cc-pCVQZlevel of theory, which allows us to derive accurate quartic centrifugaldistortion constants.The spectroscopic parameters required for predicting the rotationalspectrum are the rotational and centrifugal distortion constants.In some cases, hyperfine constants /aug-cc-pVTZ42 level of theory. SinceHONCO is not a commercial molecule, very likely it needs to be producedon-the-fly while performing the measurements, thereby exploiting ad hoc techniques, such as the flash vacuum pyrolysis,44 which, however, can lead to the concomitant formation of other (unwanted)species. Therefore, characteristic features in the rotational spectrumcan be very useful for the identification of a molecule of interest.These can be provided by electric and/or magnetic interactions, whichoccur whenever a molecule contains one or more atoms with nonzeronuclear spin I. These interactions split the rotationalenergy levels and, consequently, rotational transitions, giving riseto the so-called hyperfine structure of the rotational spectrum.41 The interaction of interest (which is the strongest one in closed-shellspecies) is the nuclear quadrupole coupling that takes place wheneverthe molecule has a quadrupolar nucleus (I ≥1) because its quadrupole moment interacts with the electric fieldgradient at the nucleus itself. In HONCO, the quadrupolar nucleusis the nitrogen atom. From a computational point of view, the predictionof nuclear quadrupole coupling constants, χij, requires the calculation of the electric field gradient atthe quadrupolar nucleusi and j refer to the inertial axes, eQ is the nuclear quadrupolemoment, and qij representsthe ij-th element of the electric field gradienttensor.41 Based on the literature on this topic , 48 (as implemented in CFOUR), allows—withinthe VPT2—the derivation of vibrational frequencies (denotedas ν) beyond the harmonic approximation. To improve the predictionof fundamental frequencies, a hybrid model is considered. This approachassumes that the differences between vibrational frequencies computedat two different levels of theory are only due to the harmonic terms,and it is well tested in the literature = ν–ω) derived from VPT2 applied to the fc-CCSD(T)/cc-pVTZanharmonic force field:The ae-CCSD(T)/cc-pCVQZharmonic force field computation, in additionto the quartic centrifugal distortion constants, also provides theharmonic frequencies (denoted as ω(ae-CC/CQZ)). The anharmonicforce field computation carried out at the fc-CCSD(T)/cc-pVTZ level,with the cubic and semidiagonal quartic force constants being obtainedby numerical differentiation of the analytic harmonic force constantsce, refs −52). In 54 where the referencewave functions were built from molecular orbital sets obtained throughstate-averaged complete active space self-consistent field (SA-CASSCF)computations.56 Singlet and triplet states werecalculated separately. The SA-CASSCF procedures were carried out byassigning equal weights to all of the considered states, i.e., thethree lowest singlet/triplet states. The molecular symmetry propertieswere explicitly used, with all calculations performed in the Cs symmetry group. The SA-CASSCFactive space consists of 14 electrons in 13 active molecular orbitals,resulting in around 37 × 103 and 64 × 103 configuration state functions (CSFs) for the singlet andtriplet states of a given symmetry species, respectively. All of thesecomputations were performed using the aug-cc-pVQZ basis set. In theMRCI procedure, the reference wave functions were built from the CASvectors by selecting only those configurations with CI coefficientslarger than 0.01. This resulted in a number of uncontracted CSFs ofaround 3.9 × 109 (5.9 × 109) and 9.4× 109 (11.7 × 109) for the singletand triplet states of A′ (A″) symmetry, respectively.CASSCF and MRCI calculations were performed using the MOLPRO 2015.1suite of ab initio programs.57Vertical excitation energieswere calculated using the internally contracted multireference configurationinteraction (MRCI) method,0 and T1 states have a monoconfigurationalcharacter, the S0-T1 transition energy has beenestimated using the CCSD(T) method, which provides more accurate resultsthan MRCI. For the purpose, we employed the fc-CCSD(T)/cc-pVQZ levelof theory to compute this energy difference, and we used this value,along with the MRCI energies for the upper triplet states, to determinethe positions  of the triplet excited states relativeto the S0 state.Since the SX̃1A′ state is a planar (Cs), quasi-linear chain, showing a trans-likeH−O−N−C moiety and around 50 MHz for both Be and Ce (∼1%).The HONCOmolecule in its iety see 1. In prele, refs ,7,8). Ths 1; see 1, while a and b being the inertial axes definingthe molecular plane and c being perpendicular toit. The approach employed for the determination of the rotationalconstants /CBS+CV+fT+pQ augmented by fc-CCSD(T)/cc-pVTZvibrationalcorrections) should provide predictions with a relative accuracy ofbetter than 0.1%,58 while for quartic centrifugaldistortion constants at the ae-CCSD(T)/cc-pCVQZ level, deviationsfrom experiment are expected to be on the order of 1%.59 While the accuracy of the computational approach employed has beenwell tested in the literature, the molecule under consideration isa challenging case because of the quasi-linearity of the NCO moiety.Using the test γ0 parameter introduced in ref , our system results tolie within the well-behaved bent molecule range. In fact, the lattergoes from γ0 = 1 to γ0 = 0.96, andfor HONCO, a value of 0.985 is obtained. Furthermore, only rotationalconstant A is actually affected, and this has a limitedimpact on the prediction of the rotational spectrum. Indeed, basedon the values of the electric dipole moment components reported in a-type and weak b-type transitions, the intensityof rotational transitions being proportional to the square of thecorresponding μ component. Therefore, the rotational spectrumof HONCO is dominated by strong aR0,1 transitionsB + C) quantity, as shown in a-type rotational transitions(|μa| = 1.75 D) are thus predicted with uncertaintiesthat mainly reflect the accuracy of the computed rotational constants B and C, which are barely affected by thequasi-linearity issue. Therefore, an accuracy of about 0.1% is alsoexpected for rotational transitions, especially for those little sufferingfrom centrifugal distortion . Theresulting simulation of the rotational spectrum in the 0–100GHz frequency range at 100 K is shown in a-type transitions , with the different Ka-components being evident.Instead, the b-type transitions are barely observablebecause of the small value of the corresponding component of the dipolemoment (|μb| = 0.47 D). The ground-state rotationalspectrum might, however, be affected by perturbations. This is a commonfeature of quasi-linear molecules and is due to a breakdown of theWatson-type asymmetric rotor Hamiltonian caused by accidental rotationallevel degeneracies. In fact, it might happen that Ka+1 levels of the ground state becomeclose in energy with the Ka levels of a low-lying, totally symmetric, vibrationally excitedstate (the v7 = 1 state in the present case). In such acase, the rotational spectrum can be modeled only for low values of Ka, but an accurate line catalogcan be usually still obtained.61 This effect was firstdescribed by Yamada,62 with the theorysubsequently detailed in ref . The results of 3 coordinate.In 65 In that spectroscopic study, the availabilityof experimental values allowed for pointing out that a posteriori correction of harmonic frequencies at the ae-CCSD(T)/cc-pCVQZ levelwith anharmonic corrections at the fc-CCSD(T)/cc-pVTZ level leadsto predictions with an average accuracy of ∼1%. However, ithas to be noted that, in the present case, the anharmonic frequencycorrection for the lowest vibrational mode (ν9) seemsto be suspiciously large /cc-pCVQZ level) are expected to provide a goodqualitative prediction for the band strength. While the ν9 fundamental has not been found significantly involved inany resonance, ν3 is affected by a Fermi resonancewith the ν4+ν7 combination band,which is responsible for the intensity transfer leading to the largecorrection noted above.As explained in d in ref ),and th–1 is very rich, even if most of the featuresare extremely weak . Above 4000 cm–1 /cc-pVTZ level (for both line position and intensity), consideringup to two quanta, is shown in 1A′ ground electronic state was evaluatedat the ae-CCSD(T)/cc-pCVQZ level of theory. Variational calculationsperformed using such a PES led to the determination of the fundamentalvibrational transitions with relative accuracy ranging between 0.2and 0.5%. In view of the limited impact expected for the approximationof computing anharmonicity at a lower level of theory, the investigationof ref . Furthermore,the barrier to linearity along the NCO bending is larger in HONCO(443 cm–1 at the fc-CCSD(T)/cc-pVTZ level; see –1 at the ae-CCSD(T)/cc-pCVQZ level; seeref . ThisiTv) from the groundto the low-lying electronic states of HONCO calculated at the MRCI/aug-cc-pVQZlevel. In these computations, as mentioned in Tv valuefor the lowest triplet state (1 3A″) has beencomputed at the fc-CCSD(T)/cc-pVQZ level. The MRCI transitionenergies for the higher triplet states were calculated by adding theappropriate energy differences to this reference value (3.73 eV).All of these transitions fall into the near-UV spectral region, withthe lowest being to the 1 3A″ state, at 3.73 eV.This particular transition results from the promotion of one electronfrom the 3a″ orbital to the 15a′ antibonding orbital.In  orbital5. Strict1A′ state at 6.66 eV, for whichthe electronic transition moment is calculated to be 1.62 D, and thesecond strongest one is to the 1 1A″ state at 4.18eV, with a transition moment of 0.36 D. All of the other transitionsare weaker. The vertical transition to the 2 1A′state results from a one-electron excitation from the bonding 12a′to the antibonding 13a′ orbital, while the transition to the1 1A″ state consists of the excitation of one electronfrom the 3a″ to the 13a′ orbital  between the two states, here computed atthe SA-CASSCF/aug-cc-pVQZ level, and Tv is the corresponding vertical excitation energy (in eV), evaluatedat the MRCI/aug-cc-pVQZ level. This equation can be immediately deducedfrom the expression of Einstein coefficients for induced and spontaneousemission  and fromthe relation τ = 1/∑mAnm. From 0 ← S1 transition, a rather short radiative lifetime of 7.84 × 10–3 μs is computed.Accordingto the symmetry selection rules for electronic transitionsof planar polyatomic molecules, all of the transitions from the groundstate to excited singlet states are possible. In HONCO, the strongestone is to the 2 ital see 5. The li0 (X 1A′) and S1 (11A″) statesof HONCO along the NO distance  compared to the HO, N=C, and C=O bonds . From –1).ance R2; 1, as obtance R2; 1. Fromt20 suggesteda HONCOquasi-linear chain among the 1:1 NH:CO2 possible compoundsoriginated from the photolysis of HN3 in solid CO2. The comparison of the fundamental frequencies from the hybrid approachwith the features reported by Milligan et al.20 does not lead to any positive match. For most of them, there isno correspondence at all. Only for one feature (1213 cm–1),20 a similar value (ν4 at 1245 cm–1) is found in 0 and S1 states of HONCO shows that this moleculeis unstable upon UV–vis irradiation. Thus, this may explainwhy it was not observed in the early experiments of Milligan et al.,20 although being the most stable isomer. Indeed,these authors irradiated HN3 trapped in a CO2 cold matrix to form HNCO2 species. Such radiation wouldhave destroyed the HONCO isomer, and solely the second most stableone, HNCO2 , is det2, NH + CO2, N + HOCO, and HCO + NO reactions,7 which are relevant for combustion and atmospheric chemistry. Concerningthe latter, the UV–vis light present in the Earth’satmosphere leads to its photodissociation at least via the S1 state. Thus, if present in this medium, HONCO is expected to liein its electronic ground state. On the other hand, its photodissociationshould participate in the modification of the atmospheric NCO radicalbudget and, more importantly, that of the OH radical. Moving to theISM, the NCO radical is the smallest species containing the backboneof the peptide bond, and, therefore, the observation of small moleculesbearing such a moiety can provide important clues on the possibleformation of amino acids in space. NCO is predicted to be abundantin dark clouds and is considered the main precursor of HNCO.68 However, the chemistry of NCO at the low temperatures of interstellarclouds has been scarcely investigated so far. In this respect, inparallel to experimental spectroscopic characterizations of HONCO,gas-phase reaction pathways leading to its formation deserve to beinvestigated.As mentioned in theIntroduction, HONCO can be formed in the complexchemistry of HCN/HNC in oxygen-rich gaseous mixtures or via the CH+ NO–1,HONCO is a rather well-behaved semirigid molecule. Therefore, ourstate-of-the-art computational investigation allowed us to provideaccurate predictions for the rotational and IR spectra, which laythe foundation for experimental characterization. The study has alsobeen extended to the lowest electronic states, which permits one toprovide the pattern of its singlet and triplet electronic states lyingin the 0–7 eV energy domain to be determined. All of thesedata are expected to be of great help in the characterization of HONCOin atmospheric, astrophysical, and combustion media. In particular,we showed that HONCO exhibits dissociation upon absorption of UV–vislight, thus producing the NCO and OH radicals. This means that HONCOis expected to participate in their budget in the Earth’s atmosphere,thus confirming its importance as an intermediate species in atmosphericchemistry.Using the first-principlesmethodology, we have characterized themost stable isomer of the  family: for the quasi-linearchain HONCO molecule, accurate structural, rotational, vibrational,and electronic properties have been determined. Despite its quasi-linearityand a torsional mode lying at ∼177 cm70 and sulfenyl thiocyanate71) play an important role in biology and in biochemistry.Therefore, on one side, a possible role of the  familyin this respect deserves to be investigated in the future, and onthe other, we suggest to extend the present accurate methodologiesto the characterization of the sulfur-substituted analogues with theaim of their identification in the laboratory and in vivo.Finally, it is noteworthy that sulfur (singly anddoubly)-substitutedanalogues of HONCO and their isomers (e.g., hypothiocyanous acid,HOSCN,


The probability of achieving a BCVA improvement of ≥15 letters at month 12 was significantly greater in the treatment-naïve group than in the previously treated group . The Cochran–Mantel–Haenszel Odds Ratio of achieving a BCVA improvement of ≥15 letters at month 12 was 3.55 . In terms of ICER, the treatment-naïve group showed cost savings of EUR 7704.2 and EUR 5994.2 for achieving an improvement in BCVA ≥ 15 letters at month 12 and at any of the measured time points, respectively. Conclusions: DEX-i was found to be more cost-effective in treatment-naïve eyes than in those previously treated with anti-VEGF. Further studies are needed to determine the most cost-effective treatment based on patient profile.Purpose: To compare the direct costs associated with the dexamethasone intravitreal implant (DEX-i) in treatment-naïve and previously treated eyes with diabetic macular edema (DME) in a real clinical setting. Methods: Retrospective and single-center study conducted in a real clinical scenario. Consecutive DME patients, either naïve or previously treated with vascular endothelial growth factor inhibitors (anti-VEGF), who received treatment with one or more DEX-i between May 2015 and December 2020, and who were followed-up for a minimum of 12 months, were included in the study. The cost analysis was performed from the perspective of the Andalusian Regional Healthcare Service. The primary effectiveness endpoint was the probability of achieving an improvement in best-corrected visual acuity (BCVA) ≥ 15 ETDRS letters after 1 year of treatment. The incremental cost-effectiveness ratio (ICER) of different improvements in BCVA was calculated. Results: Forty-nine eyes, twenty-eight (57.1%) eyes from the treatment-naïve group and twenty-one (42.9%) from the previously treated group, were included in the analysis. The total cost of one year of treatment was significantly lower in the treatment-naïve eyes than in the previously treated eyes [Hodges-Lehmann median difference: EUR 819.1; 95% confidence interval (CI): EUR 786.9 to EUR 1572.8;  Recent years have seen major socio-demographic changes that have inevitably been challenges to health systems. While these changes started initially in high-income countries, it is now low- and middle-income countries that are experiencing these problems. It is estimated that by 2050, approximately 1 in 6 people in the world will be over the age of 65, compared to 1 in 11 in 2019 , mean [95% confidence interval (95% CI)] or median [Interquartile range (IqR)] were used as appropriate.The distribution of quantitative variables was assessed using the D’Agostino–Pearson test.The Mann–Whitney U test was used to assess baseline quantitative variables between naive eyes and previously treated eyes.The Cochran–Mantel–Haenszel test was used to assess the probability of achieving a BCVA improvement ≥ 15 letters (at any measured time point and at month 12) or ≥10 letters (at any measured time point and at month 12) with study group as the grouping variable and lens status as the factor variable.Analysis of covariance (ANCOVA) was used to assess variation in BCVA and CRT between study groups. The model included “Group” (treatment-naïve versus previously treated) as a factor and age, DME duration and lens status as covariates.Forty-nine eyes were included in the analysis: twenty-eight (57.1%) eyes in the treatment-naïve group and twenty-one (42.9%) in the previously treated group.Baseline demographic and clinical characteristics are summarized in p < 0.0001).The total cost of one year of treatment was significantly lower in the treatment-naïve eyes  than in the previously treated eyes  .The probability of achieving an improvement of BCVA ≥ 15 letters at month 12 was significantly higher in the treatment-naïve group  than in the previously treated group    .The CER for an improvement of ≥15 letters in BCVA at 12 months was EUR 4411.9 in the treatment-naïve group and EUR 12,116.1 in the previously treated group. All measurements in the treatment-naïve group showed lower CERs .Regarding ICER, the treatment-naïve group achieved cost savings of EUR 7704.2 and EUR 5994.2 at month 12 and ≥15 letters in BCVA improvement at each measured time point, respectively .p < 0.001 in each, as compared to baseline values. Friedman test).The median number of ETDRS letters gained after treatment with the DEX-i was significantly higher in the treatment-naïve group than in the previously treated group at months 2, 4, 8 and 12 A. Howevep < 0.001 versus baseline. Friedman test)  B.p = 0.0134; p = 0.0060; and p = 0.0092, respectively) (p = 0.0463), there were no significant differences in CRT reduction between treatment-naïve and previously treated eyes  . With thted eyes .p = 0.0124).The median number (IqR) of DEX-i administered throughout the study was 2.0 (1.5 to 2.0) in the treatment-naïve and 2.0 (2.0 to 3.0) in the previously treated group , nor the interaction between both variables (p = 0.825).Regarding the cost analysis in patients with subretinal fluid, in the comparison of the cost adjusted by group and SRF, the presence of SRF was not relevant , nor the interaction between both variables (p = 0.825).Multiple hypotheses relate certain anatomical characteristics with a better response to DEX-I implants, such as the presence of subretinal fluid. In a previous analysis by the authors , the preOn the other hand, the design of this study did not have the objective of analyzing these anatomical characteristics or the cost in relation to them.After adjusting for different covariates, visual acuity improvement was significantly greater in treatment-naïve eyes than in the previously treated eyes at months 2, 8 and 12. These findings are consistent with the results of previously published studies that indicated a greater improvement in BCVA in naïve compared with non-naïve DME eyes ,33,34,35p < 0.0001, each, respectively), which is consistent with previous published studies [Regarding CRT, there was a significant reduction at month 12 in the difference from baseline in naive and previously treated eyes , patient transport and other ancillary costs to determine economic parameters.DEX-i proved to be more cost-effective in untreated eyes than in eyes previously treated with anti-VEGF. The costs of untreated patients were significantly lower in all aspects except for the costs associated with cataract surgery. In conclusion, this study demonstrates that DEX-i is effective in treating eyes with DME even in difficult cases where previous anti-VEGF treatment has failed, although functional outcomes were better in untreated eyes. However, this study did not compare the cost-effectiveness of DEX-i versus anti-VEGF in treatment-naïve eyes.VEGF inhibitors are currently the first-line treatment for DME patients. However, many patients respond inadequately. Further studies, mainly prospective and multicenter, are therefore warranted to determine the most cost-effective treatment according to the patient profile.


BackgroundMany international studies have covered the predictors of prostate cancer, but there is limited information pertaining to Likert 3 MRI scores and the diagnosis of clinically significant prostate cancer (cs-PCa). Therefore, this study aimed to assess the detection rate of significant prostate cancer in men with a Likert 3 score multiparametric MRI (mp-MRI) and the predictive value of prostate-specific antigen (PSA) density in detecting significant prostate cancer.MethodsThis is a retrospective analysis of patients referred for suspected confined prostate cancer. Inclusion criteria were patients with prostate mp-MRI score of Likert 3 and a prostate biopsy performed. Exclusion criteria included grossly abnormal feeling prostate, no biopsy performed, and an mp-MRI score (Prostate Imaging-Reporting and Data System/Likert) of 1, 2, 4, and 5. cs-PCa was defined as ≥ Gleason 3+4 prostate cancer. PSA density (PSAD) was calculated from MRI estimation of prostate volume. PSAD and histology results were subjected to receiver operating characteristic (ROC) curve analysis with the intention to assess the detection rate of significant prostate cancer in men with Likert 3 mp-MRI and the predictive value of PSAD in detecting significant prostate cancer.ResultsA total of 819 eligible men had a pre-biopsy mp-MRI scan taken between October 2019 and March 2022. A total of 177 men  were Likert 3 positive, and 31 did not proceed to take prostate biopsies. A total of 146 patients were included in the study. The median PSAD was 0.19 in men with cs-PCa. Prostate cancer was detected in 42 men , of which 27 (18.5%) had a Gleason 3+3 prostate cancer and 15 (10.3%) had Gleason ≥ 3+4 prostate cancer. Therefore, 35.7% (n = 42) of biopsy-positive men with Likert 3 mp-MRI had cs-PCa.The ROC curve analysis confirms that PSAD is a predictor of cs-PCa. The optimal PSAD threshold was 0.16 (95% CI: 0.14-0.19), which gives an accuracy of 0.7371, a sensitivity of 0.7333, and a specificity of 0.7375.ConclusionThe specificity of PSAD is arguably insufficient for it to stand alone as a decision-making tool when counseling men with equivocal mp-MRI on whether or not to undergo prostate biopsy. A predictive model will need to incorporate other independent risk factors. These may include lesion size, multiplicity, location of lesion(s), and age. Prostate cancer is the second most commonly diagnosed cancer in men , with a A prostate MRI stratifies patients on a scale of 1 to 5 using Prostate Imaging-Reporting and Data System  , with a The recommendation by NICE is supported by all prostate cancer and cs-PCa biopsy detection rates of 19-38.6% and 11-45%, respectively -18 withiPatient populationThis is a retrospective study. We performed a retrospective analysis of the hospital database at Glan Clwyd Hospital, Wales to identify men referred for suspected organ-confined prostate cancer between the dates of October 2019 and March 2022. We identified 819 men who had pre-biopsy mp-MRI of the prostate performed. Out of these, 642 (78.4%) men had mp-MRI scored at Likert 1, 2, 4, or 5 and were excluded. A total of 177 (21.6%) men had mp-MRI scored at Likert 3 and 31 men from this cohort did not have prostate biopsies and were also excluded. A total of 146 men were included in the study. The Likert scale was chosen as it adheres to most of the reporting structure in PI-RADS V2 with additional variables like radiologist experience and clinical risk factors. It is recommended in the UK by NICE for use in practice and has evidence supporting better detection of cs-PCa over PI-RADS scoring.Prostate cancercs-PCa was defined as International Society of Urological Pathology (ISUP) group 2 (Gleason score 3+4) prostate cancer and above.MRIAll the men had an mp-MRI of the prostate prior to biopsy with a 1.5T Siemens Sola machine . The protocol included high-resolution T2-weighted (T2WI) axial/coronal/sagittal, T1 axial wide field pelvis, T1 coronal wide field abdomen-pelvis, T2 axial wide field upper abdomen, diffusion-weighted (DWI) prostate, and dynamic contrast-enhanced (DCE) prostate. The MRI images were reviewed by three experienced uro-radiologists. PSAD was calculated from MRI estimation of prostate volume and included in the report.Prostate biopsyTransrectal ultrasound (TRUS)-guided biopsies were performed if the lesions were large or multiple and located in the peripheral zone on mp-MRI. Five systematic biopsies were taken from each lobe of the prostate.Transperineal (TP) biopsies under TRUS guidance were performed if the lesion was small and solitary or located in the anterior zone. Targeted biopsies of the lesion as well as systematic biopsies from each lobe were taken. Biopsy samples were labeled to indicate the region of the prostate sampled, and reported accordingly.Images and pathology were reviewed at a multidisciplinary meeting with reporting uro-radiologists, pathologists, and urologists in attendance.Statistical analysisThe factor evaluated as a predictor of cs-PCa was PSAD. To determine the optimal PSAD threshold for detecting men with cs-PCa, a receiver operating characteristic (ROC) analysis was conducted using the R statistical software  and the cut-off point package . The thrPatient characteristicsThe median age, PSA, and prostate volume as well as the range of each variable for our patient cohort Table  was 65 yMRIAll men had the same mp-MRI protocol performed on a 1.5T Siemens Sola machine. We stratified patients with respect to their Likert scores below Table .Biopsy techniqueA total of 146 men had prostate biopsies, of which 118 (80.8%) had TRUS biopsies while 28 (19.2%) had TP ultrasound biopsies performed.Biopsy histologyTable PSAD evaluation and prediction of cs-PCaThe optimal threshold for identifying cs-PCa using PSAD was found to be 0.16 (95% CI: 0.14-0.19). This threshold gives an accuracy of 0.7371, a sensitivity of 0.7333, and a specificity of 0.7375, as seen below  analysis with a sensitivity of 0.73 and a specificity of 0.74 at an optimized PSAD threshold of 0.16. This threshold is similar to several studies ,23,25,27LimitationsThe limitations of this study include but are not limited to the fact that retrospective analysis of data lends itself to bias, where results could change in prospective follow-up. A relatively small sample size is within this study, as it is expected that the population of mp-MRI scored as Likert 3 will be small when reported by experienced uro-radiologists. Our study was done in a single center with multiple clinicians performing prostate biopsies. The percentage of Gleason 4 in patients in ISUP group 2 was unfortunately not assessed in this study, as that would complicate analysis.There was no option to perform fusion biopsies in our hospital. We appreciate MRI/TRUS-fusion targeted biopsies have a higher diagnostic yield compared with systematic TRUS-guided biopsies. However, studies comparing cognitively targeted TRUS biopsies with fusion biopsies have yielded mixed results.We also utilized 1.5T MRI images. 3T mp-MRI machines do produce better images, but we found no evidence that the reporting outcomes from 1.5T mp-MRI are inferior to 3T MRI machines in mp-MRI of the prostate, therefore warranting no significant concern. Various nomograms have been proposed, combining multiple clinical parameters, while we only studied PSA. We agree with this sentiment, but none of the proposed nomograms have been validated and widely agreed upon either nationally or internationally. As authors, we chose PSAD because this is currently the most widely used “objective” surrogate marker in deciding if a patient with an mp-MRI with a score of Likert 3 or PI-RADS 3 should proceed to prostate biopsy (see NICE UK recommendation). This study contributes to the body of evidence that PSAD alone is inadequate.The use of mp-MRI has lived up to the claims of the PROMIS study, and 35% of men in this study safely avoided prostate biopsy. The use of PSAD, calculated from mp-MRI, has gained traction as a reasonably predictive decision-making tool when counseling men with equivocal mp-MRI on whether or not to undergo prostate biopsy. However, this study has demonstrated that the specificity of PSAD with regards to cs-PCA in an equivocal mp-MRI is arguably insufficient for it to be a lone surrogate factor in reaching a decision on prostate biopsy as a stand-alone investigation. There is still a need for a better predictive model to guide the recommendation of prostate biopsy for men who underwent an mp-MRI and while PSAD will certainly be a significant variable in determining this improved predictive model, other independent factors are required and may be implemented in the future. These may include lesion size, number of lesions, location of lesion(s), and age.


Interleukin-6 (IL-6) is involved in inflammation and has a significant role in chronic lymphocytic leukemia (CLL) progression. Accordingly, IL-6 level may increase in CLL-affected patients compared to healthy individuals. The -174G>C single nucleotide polymorphism (SNP) in IL-6 promoter region has been related to differences in IL-6 transcription. Therefore, we investigated the possible association of IL-6 polymorphism with CLL. IL-6 gene and studied its possible relationship with CLL in affected patients and in healthy controls using Amplification Refractory Mutation System- polymerase chain reaction genotyping method. IL-6 plasma level was measured in both studied groups.We examined the -174G>C SNP in IL-6 gene was not associated with CLL. Furthermore, there were no significant differences in the distribution of allele and genotype frequencies between the CLL-affected patients and the controls (P>0.05). According to the results, IL-6 mean plasma concentration was increased significantly in the CLL patients compared to the controls. However, 174G>C genotype of the IL-6 gene could not be considered a risk factor for CLL. Larger prospective studies should be performed to confirm our results.Our study showed that -174G>C SNP in promotor of  The B-cell chronic lymphocytic leukemia (B-CLL) cells growth is associated with exogenous growth factors (in vitro) . Cytokin Interleukin-6 (IL-6) as a multifactorial cytokine may have a role in the severity and progression of numerous types of cancers. Various observational studies have proposed that circulating IL-6 can describe inter-individual variability in cancer predisposition . IL-6 gene were investigated  is produced by immune cells, adipose tissue, and cardiovascular components. This cytokine primarily functions in the progression of inflammation, as well as functioning as an endogenous pyrogen in patients with infection , 6. SeveIL-6 gene, its possible susceptibility to CLL, and also its probable effect on IL-6 plasma levels. To this end, we comparatively evaluated the -174G/C SNP of IL-6 in Iranian CLL-affected patients and the controls.  Considering the above-mentioned, we aimed to evaluate the −174G>C single-nucleotide polymorphism (SNP) in Demographic Data and Blood SamplingIL-6 gene, 35 CLL patients and 34 healthy controls from the hospitals affiliated to Urmia University of Medical Sciences, Iran, were included in this case-control study. The study protocol was reviewed and approved by Urmia University of Medical Sciences (Code of Ethics: IR.UMSU.REC. 1395.272).In order to evaluate the -174G/C SNP in the promoter region of Patients were aged 27-64 years with a history of confirmed CLL who had referred to the educational hospitals of Urmia University of Medical Sciences, between January and September 2019 and were recruited as the patient group. CLL patients with other disorders were excluded from this study. Demographic information  of the subjects in both study and control groups were collected.Based on an oncologist diagnosis, 35 CLL patients and 34 healthy controls were considered for further analysis. Then in the CLL patients, CD19, CD5, and CD23 markers were evaluated using immunophenotyping  Table 1Table 1. Genomic DNA Extraction and ARMS-PCR for −174 G>C SNP DetectionARMS-PCR) genotyping method was employed  obtained from the patients and controls were subjected for the genomic DNA extraction using the manufacturer’s protocol . DNA was isolated from the peripheral blood leukocytes to identify the −174G>C gene polymorphism. The extracted DNA was visualized on agarose gel 1% . To dete2O. The PCR amplification was performed as follows: initial denaturation at 94°C for 10 min, 40 cycles of denaturation at 94°C for 45 s, annealing at 58°C for 45 s, and extension at 72°C for 45 s, and final extension at 72°C for 7 min.  ARMS-PCR reaction was carried out in a total 20-μL volume comprising DNA sample, forward and reverse primers, dNTP (0.3 μL), MgCl2, Pfu enzyme, and ddHThe products of ARMS-PCR were visualized on Agarose gel 1%, and were compared to the 100-bp DNA Ladder (Thermo Scientific). Then, the odds ratios were utilized for investigating the relationships between IL-6 genetic polymorphism and CLL .Statistical Analysis Statistical analyses were performed using the IBM SPSS software 22  and applying the Pearson Chi-Square test. Indeed, Chi-square test was utilized to compare the genotype and allele frequencies of the −174G>C SNP between the cases and controls. A P-value <0.05 was considered as the significance level. Homozygosis and heterozygosis were directly counted in the control and patient groupsIL-6 gene, genomic DNA was extracted from blood samples of the patients and controls and evaluated by ARMS-PCR method. The quality of extracted DNA was confirmed by Agarose gel 1% (P>0.05).  To assess the 174G>C polymorphism in the promotor region of e gel 1% . The imme gel 1% , in subgAfter performing ARMS-PCR, the PCR products were visualized on Agarose gel 1% .P>0.05) in the frequency of IL-6, -174 GG genotype and G allele (P>0.05) between the CLL group and the controls. Furthermore, there was no significant increase in the distribution of IL-6 GG genotypes, and an insignificant reduction was observed in the frequency of GC genotype (P >0.05) between the CLL patients and the control subjects .The mean serum concentration of IL-6 was increased significantly in the CLL cases compared to the controls  to G (guanine) transition]  between the healthy controls and CLL patients. Additionally, the subgroup analysis showed that -174G>C polymorphism distribution was not significantly different between the two studied groups. Consequently, we conducted this case-control study to evaluate the relationship between the SNP in promoter region of et al., IL-6 plasma concentrations in the B-CLL patients were increased compared to the healthy subjects, corroborating our results. The authors also evaluated the polymorphism in the IL-6 gene. They indicated that the allele frequencies of the studied genes were comparable in the controls and patients. They indicated that some mechanisms except for allelic imbalance of their genes were the main reasons for the profiles of cytokine detected in this disease , 27 subjects with gammopathy of undetermined significance and 129 healthy controls were included. The results showed that the tibility . IL-6 gene was not related to CLL. There was no difference in the distribution of −174G>C SNP in the CLL cases compared with the controls (P>0.05). Distribution of the GG genotype was not different between the CLL and healthy groups (P>0.05). In addition, the IL-6 GC genotype was not linked to the CLL. We did not find any exact similar study to compare with those found in our study. According to our results, the mean plasma IL-6 level was significantly increased in the CLL patients compared to the healthy controls. Moreover, the −174G>C genotype of IL-6 gene and CLL. These findings suggested that predisposition of genetic differences in the IL-6 gene might not be associated with the increased risk of CLL in this studied population. The IL-6 polymorphisms may not have a role in CLL susceptibility in the studied patients. Overall, our preliminary study displayed that G allele and GG genotype of IL-6 (-174G/C) could not be considered as CLL-related risk factors. Larger prospective studies should be conducted to confirm these results.We evaluated frequencies of the −174G>C SNP and its possible effect on the levels of IL-6 as a pro-inflammatory cytokine. Our results revealed that there was no association between the −174G>C polymorphism in the Urmia University of Medical Sciences.This study supported financially by None.


Biomarkers of different stages and grades of bladder cancer (BC) are important in clinical work. The objective of our study was to investigate new biomarkers of early-stage BC with liquid chromatography-high resolution mass spectrometry (LC-HRMS) using serum samples.A total of 215 cases were included in our study, including 109 healthy adults as the control group and 106 non-muscle invasive bladder cancer (NMIBC) patients as the NMIBC group. Serum samples were collected from BC patients in the early stage, called NMIBC, and healthy people before surgery. We used LC-HRMS to distinguish the NMIBC group from the control group and the low-grade NMIBC group from the control group.An apparent difference between the NMIBC group and the control group was visualized by unsupervised principal component analysis (PCA). Metabolite panels for 16-hydroxy-10-oxohexadecanoic acid, PGF2a ethanolamide, sulfoglycolithocholate, and threoninyl-alanine were used to distinguish the two groups. The area under the curve (AUC) of the panels was 0.985, and the sensitivity and specificity were 98.63% and 98.59%, respectively. To distinguish the low-grade NMIBC group from the control group, serum metabolic profiling differences between the low-grade NMIBC group and control group samples were also analyzed. Metabolite panels of L-octanoylcarnitine, PGF2a ethanolamide, and threoninyl-alanine showed good discrimination performance. The AUC of the panels was 0.999, and the sensitivity and specificity were 97.8% and 100%, respectively.Metabolomics analysis of serum samples can distinguish the NMIBC group from the control group, particularly the early-stage low-grade NMIBC group. The incidence of bladder cancer (Bca) has increased in recent decades. Bca is still a worldwide problem in people over the age of 50, and the incidence differs among races, with a lower morbidity but a poor survival rate in African Americans . The morin situ (CIS). Because of these problems, biomarkers and metabolic products with high sensitivity and specificity are necessary.The current diagnosis of Bca includes cystoscopy, urinary cytology, computed tomography (CT), ultrasound (US), and magnetic resonance imaging (MRI). Bladder tumor antigen (BTA) and nuclear matrix protein 22 (NMP-22) are traditional biomarkers that have low sensitivity and specificity. Urinary cytology has low sensitivity, and cystoscopy is expensive and invasive. Tumors are classified into two different types: non-muscle invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Transurethral bladder tumor resection (TURBT) is the main method for treating Bca in its early stages. However, the sensitivity and specificity of cystoscopy and urinary cytology are low, and radiological examination has difficulty detecting some types of NMIBC in the early stage, for instance, papillary tumors limited to the mucosa and carcinoma As individual risk prediction markers, metabolites have been identified with disease signatures . In earlIn this study, we further studied serum metabolic profiling in the diagnosis of NMIBC. Furthermore, we discovered biomarker panels that could distinguish between NMIBC patients and controls, low-grade NMIBC patients and controls, and high-grade NMIBC patients and controls.g for 10 min. After that, the supernatant was dried and stored. Before transferring the samples to an autosampler, 10 kDa molecular weight cut-off ultracentrifugation filters  were used for small-protein depletion. The dried powder that remained was reconstituted in 200 µl of 2% acetonitrile. This research was approved by the Institutional Review Board of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.All patients and their families provided informed consent before the study. According to the 2004 classification criteria of the World Health Organization/International Society of Urological Pathology, papillary urothelial proliferation of low-malignant potential (PUNLMP), low-grade papillary carcinoma, and high-grade papillary carcinoma NMIBC patients were collected. The serum samples were all collected before the TURBT operation and centrifuged within 6 hours, and the supernatants were stored at −80°C after isolation and aliquoting. The process was as follows: 200 µl of serum was mixed with 200 µl of acetonitrile and vortexed for 30 s. Then, the samples were centrifuged at 14,000×A Waters ACQUITY H-class LC system coupled with an LTQ-Orbitrap mass spectrometer  was used. A Waters HSS C18 column  was used to perform metabolite separation at a flow rate of 0.5 ml/min using a 17-minute gradient. The gradient was set at 1–3 min, 2%–55% solvent B; 0–1 min, 2% solvent B; 3–8 min, 55%–100% solvent B; 8–12 min, 100% solvent B; 12–12.1 min, 100–2% solvent B; and 12.1–17 min, 2% solvent B. The mobile phases were 0.1% formic acid in H2O (A) and acetonitrile (B). The column temperature was 50°C. The mass scan ranged from 100 to 1,000 m/z. The MS1 analysis resolution was set at 60 K, and the MS2 resolution was 15 K. The automatic gain control was set as 1 × 106 with a maximum injection time (IT) of 100 ms. The MS2 automatic gain control was set as 5 × 105, and the maximum IT was 50 ms. Metabolites were dissociated by higher-energy collisional dissociation (HCD) fragmentation mode with an optimal collision energy of 20, 40, 60, or 80.http://www.metaboanalyst.ca), including missing value estimation, log2 transformation, and Pareto scaling. Variables missing in 50% or greater of all samples were removed from further statistical analysis, and the other missing values were filled using the KNN method. Pattern recognition analysis  was performed using SIMCA 14.0  software. The variable importance for projection (VIP) value obtained from OPLS-DA was used for differential metabolite selection. Nonparametric tests (Wilcoxon rank sum test) were used to evaluate the significance of variables. False discovery rate (FDR) correction (Benjamini method) was used to estimate the chance of false positives and correct for multiple hypothesis testing. Differential metabolites were selected according to the following criteria: (1) VIP value >1; (2) adjusted p-value <0.05. A ROC curve was constructed based on differential metabolites using a logistic regression algorithm.Raw data files were processed by Progenesis QI  software. The mass list data file exported from QI was further processed by MetaboAnalyst 5.0  group and the control group, as well as the low- and high-grade NMIBC group and the control group. So, TNM stage and pathological type did not influence the results of the study. The detailed general information is listed in Serum metabolic profiling differences between the control group and NMIBC samples were analysed. Potential biomarkers were discovered to distinguish NMIBC. Additionally, to perform early detection of Bca, serum metabolic profiling differences between the control group and early-stage low-grade cases were also analyzed. The workflow is shown below Figure 1This cohort of samples was analyzed in one center, and quality control (QC) was important to limit bias. Almost every 5 days, all samples are analyzed. The QC samples were prepared using a total of 80 representative samples mixed in equal aliquots, and they were injected every 10 samples. PCA showed that QC samples clustered slightly. The QC results showed that biological variations are the key to distinguishing group differences  values of the 33 metabolites were greater than 0.8, which indicates their potential value in clinical diagnosis. We selected the potential biomarker panel based on its biological significance and used K-means (KM) clustering to detect features with similar behavior to help reduce the redundancy in biomarkers . K-means (KM) clustering was performed by MetaboAnalyst 5.0 (“Biomarker discovery” module). Finally, metabolites of 16-hydroxy-10-oxohexadecanoic acid, PGF2a ethanolamide, sulfoglycolithocholate, and threoninyl-alanine were used to construct a predictive model for NMIBC Table S2Exploring potential biomarkers for the early differential diagnosis of the low-grade NMIBC group and the healthy group is very important in clinical work and treatment. PCA and the OPLS-DA model were used again to discriminate between the control group and the low-grade NMIBC group  serum levels . To our Yasushi et al.  exploredOur results further confirmed that 16-hydroxy-10-oxohexadecanoic acid was only sensitive to low-grade bladder cancer, and the function of 16-hydroxy-10-oxohexadecanoic acid was limited in the detection of bladder cancer at an early stage. Liu et al.  reportedL-Octanoylcarnitine was reported and proven by Zoni et al.  showed aWe also determined that distinguishing between the high-grade NMIBC group and the low-grade NMIBC group is very important in clinical work and treatment. PCA was also performed, and the results showed that there were no significant differences between the high-grade NMIBC group and the low-grade NMIBC group. The following reasons may explain the results (1): the sample size was small, which may produce bias, or (2) the tumor cytological structure was too like distinguish them from each other in the two groups. There are also some limitations in our study: (1) In our previous study, urinary metabolites had been analyzed in NMIBC patients, and dopamine 4-sulfate, MG00/1846Z,9Z,12Z,15Z/00, aspartyl-histidine, and tyrosyl-methionine were found to distinguish NMIBC patients, and 3-hydroxy-cis-5-tetradecenoylcarnitine, 6-ketoestriol, beta-cortolone, tetrahydrocorticosterone, and heptylmalonic acid were found to distinguish low-grade NMIBC patients . FurtherMetabolomics analysis of serum samples was able to distinguish the NMIBC group from the control group and the low-grade NMIBC group from the control group. Further studies are required to find more biomarkers based on larger samples and multiple centers.The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.This research was approved by the Institutional Review Board of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. All patients and their families were provided informed consent before the study.YZ: Manuscript writing/editing, Data collection or management. WS: Protocol/project development. ZJ: Protocol/project development. XL: Data analysis. YQ: Data collection or management. All authors contributed to the article and approved the submitted version.